The goals of the proposed study are to test the hypotheses that muscarinic acetylcholine receptor (mAChR) subtypes are differentially expressed at unique pre- and post-synaptic sites in key hippocampal circuits and that their expression is altered in Alzheimer's disease (AD). Muscarinic cholinergic transmission in hippocampus is important in learning, memory and attention, and loss of cholinergic input may contribute to cognitive dysfunction in AD. Five mAChR subtypes (m1-m5), encoded by distinct genes, are potentially valuable targets for new cholinergic therapies. Differential expression of ml-m5 receptors in hippocampus suggests a greater complexity in cholinergic modulation of neurotransmission than was previously postulated. A diversity of pre- and post-synaptic cholinergic actions may result from differential cellular/subcellular expression of mAChR subtypes in hippocampus. However, the roles of ml-m5 are poorly understood because the available pharmacological tools do not provide molecular specificity or the spatial resolution necessary to localize the receptors to pre- vs. postsynaptic sites. Determination of the detailed localization of mAChR subtypes in memory circuits and their alterations in AD are necessary first steps toward understanding the molecular basis for cholinergic modulatory actions and for realization of new therapeutic strategies. A panel of mAChR subtype-specific antibodies has been previously developed which has enabled the molecular specificity and high spatial resolution necessary to characterize the synaptic localization of the individual mAChR proteins in brain. In preliminary studies in rat hippocampus, ml-m4 proteins are localized with strikingly different regional, laminar, and cellular distributions in the light microscope. The first aim of these studies will use immuno-electron microscopy to test the hypothesis that mAChR subtypes have distinct pre- and postsynaptic distributions. The second aim will use neuroanatomical tracing techniques (anterograde degeneration and anterograde tracers) combined with immuno-electron microscopy to delineate the pre- and postsynaptic receptor proteins at identified Schaffer collateral synapses in CAL. and at identified perforant pathway and commissural synapses in dentate gyrus. The mAChR proteins localized at these synapses will be visualized using immunoperoxidase and immunogold techniques. The final aim will use light microscopic immunocytochemistry to test the hypotheses that mAChR proteins are expressed in a similar distribution in human and rat hippocampus, and that recently determined alterations in the levels of individual mAChR proteins in AD are due to regional-, laminar-, and cell-specific alterations in their distributions in AD compared to age-matched controls. The results will provide novel insights into the roles of the molecularly distinct mAChR subtypes in rat and human hippocampus, their alterations in AD, and their potential value in developing new pharmacological therapies.